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  • Periodic Report Summary 3 - DESIRE (Development and Epilepsy - Strategies for Innovative Research to improve diagnosis, prevention and treatment in children with difficult to treat Epilepsy)

Periodic Report Summary 3 - DESIRE (Development and Epilepsy - Strategies for Innovative Research to improve diagnosis, prevention and treatment in children with difficult to treat Epilepsy)

Project Context and Objectives:
Epilepsy and epileptiform disorders impose a major medical and socio-economic problem due to their high prevalence.

DESIRE focuses on epileptogenic developmental disorders EDD, i.e. early onset epilepsies whose origin is closely related to developmental brain processes. A major cause of EDD are malformations of cortical development (MCD), either macroscopic or subtle. EDD are often manifested as epileptic encephalopathies (EE), i.e. conditions in which epileptic activity itself may contribute to severe cognitive and behavioural impairments. EDD are the most frequent drug-resistant pediatric epilepsies carrying a lifelong perspective of disability and reduced quality of life.
Updated and comprehensive 2010 estimates for European countries (Olesen et al, 2012; Eur J Neurol, 19:155-62) revealed an annual economic cost of about 13.8 billion euros for epilepsy, and drug-resistant epilepsies would consume the most significant portion.
Although EDD collectively represent a major medical and socio-economic burden, their molecular diagnosis, pathogenic mechanisms (PM) and rationale treatment are poorly understood.
Lack of knowledge and of targeted therapeutic interventions creates an enormous burden to families confronted with such disorders.
As many patients with EDD develop drug resistance and cognitive decline, improved diagnostic strategies are mandatory for personalized therapy.

Specific objectives of DESIRE are to advance the state of the art with respect to:
(1) the genetic and epigenetic causes and PM of EDD, particularly epileptogenic MCD, to elucidate molecular networks and disrupted protein complexes and search for common bases for these apparently heterogeneous disorders.
(2) the diagnostic tools (biomarkers) and protocols through the study of a unique and well-characterized cohort of children to provide standardized diagnosis for patient stratification and research across Europe.
(3) treatment of EDD using randomized, multidisciplinary clinical protocols and testing preclinical strategies in experimental models to also address novel preventative strategies.

Project Results:
In the framework of Workpackage 1, which aims at Identifying genetic causes and pathophysiological mechanisms of epileptogenic brain malformations, we enrolled 279 additional patients/families with focal or diffuse malformations of cortical development (mainly focal cortical dysplasia, hemimegalencephaly periventricular nodular heterotopia and polymicrogyria) and 390 additional patients/families with epileptic encephalopathies with seemingly non-lesional origin. Using NGS panels designed at P1 UNIFI, we screened 349 patients with seemingly non-lesional epilepsy (EPI panel), 237 patients with malformations of cortical development (MCD panel), and 150 patients with phenotypic features suggesting MTOR pathway genes involvement (MTOR panel), identifying causative genes in 20%, 14.2%, and 8.8% of patients, respectively. Through genetic screening and patients phenotypic characterization, we demonstrated that GABRA1 gene should be considered in patients with generalised EE, especially those with infantile-onset, prominent tonic-clonic and myoclonic seizures, generalized spike-and-wave seizures, and a photoparoxysmal response on the EEG. We also demonstrated that hyperkinetic–dyskinetic movement disorder should be considered a distinctive feature of the FOXG1-related phenotype and that in children with epileptic encephalopathy with migrating partial seizures, concomitant multisystem involvement should prompt investigations for Congenital Disorders of Glycosylation (CDG). Combining massive parallel sequencing of genes belonging to the MTOR pathway, WES, and functional studies, we contributed to demonstrate that MTOR gene mutations are associated with a spectrum of brain overgrowth phenotypes extending from FCD type 2a to diffuse megalencephaly and distinguished by different mutations and levels of mosaicism. We also substantiated the link between mosaic mutations of MTOR and pigmentary mosaicism in skin. In addition, we proved that PIK3CA-related overgrowth comprises a discontinuous spectrum of disorders that correlate with the severity and distribution of mutations. Using the same approach, we demonstrated that NEDD4L is a novel causative gene for periventricular nodular heterotopia and showed that mutations altering the HECT domain of this gene, as well as an excess of NEDD4L product, lead to MCD and neurodevelopmental defects. Through functional studies, we showed that alterations in NEDD4L caused disruption of the interplay between ubiquitination mediated by NEDD4L itself and PI3K/AKT/mTOR signalling pathway. These results revealed a potential novel disease-causing molecular mechanism in which missense mutations might lead to a constitutive active state and loss of the mutant protein, but with functional consequences that are different from constitutive haploinsufficiency.
Performing epigenetic studies, we showed methylation signatures to be highly distinct in seizure versus non-seizure control samples and non-FCD seizure groups (i.e. TLE-HS). In particular, we identified a significant anti-correlation specifically at promoters, i.e. hypermethylation was associated with reduced gene expression whereas hypomethylation was primarily found in upregulated genes and that genes that are hypermethylated and downregulated in FCD type IIB patients shared specific binding motifs. Among them, H3K27me3 signatures primarily help to distinguish FCD type IIb samples from FCD type IIa, non-seizure controls and TLE-HS. We also identified significant differences between FCD Type-IIA and FCD Type-IIB for cg04221606 (P = 6.8 x 10-12) located in a CGI shore flanking the TSS of AKAP12, an effector of the rapid epithelial barrier reconstruction after injury and cg09762612 (P = 7.5 x 10-8), which is located in a CGI in exon two of the LT3 Interacting Zinc Finger 1 (FIZ1) on 19q13.42. We also demonstrated that the majority of the meQTLs in brain tissues are also detectable in whole blood cells, providing a valuable key to select accessible epigenetic biomarkers for brain disorders in whole blood cells.
Analysing brains of FlnAEmx1-Cre mice, we identified a clear enlargement of the lateral ventricles together with morphological alterations of the hippocampal shape, and a discrete dense cellular mass suggestive of a rather local perturbation of neuronal migration reminiscent of the nodular heterotopia reported in FlnA patients. We did not find gross alterations in cortical structure as well as cortical layering. Neurons in brains of FlnAEmx1-Cre mice exhibited a slight but significant increase in their dendrites’ outgrowth when compared with control neurons. This increase involved specifically the arborisation of the basal dendrites component.
The functional studies we performed to characterize Eml1, a giant heterotopia related gene recently identified in the framework of Workpackage 1 activities, revealed that this gene has a cell cycle-dependent localization, enriched along spindle microtubules. In agreement with these observations, we demonstrated that the brain of HeCo mouse (a spontaneous model of Eml1 inactivation) shows changed in spindle orientations, which may contribute to progenitor detachment. Eml1 mutant mice have also significantly increased spindle lengths compared to wild-type, and mutant cells are also significantly larger than control cells. Furthermore, microtubule dynamics are perturbed in Eml1 mutant cells, which may contribute to the spindle length abnormalities. Silencing of DLGAP4, a candidate gene for giant heterotopia with polymicrogyria, or periventricular nodular heterotopia and partial agenesis of corpus callosum, demonstrated that this gene is associated with cell disorganization, suggesting a possible role in maintaining cell polarity at the ventricular lining.
The studies we performed in the ferret showed that the Outer Subventricular Zone (OSVZ) is a key element of cortical development in large and gyrencephalic cortices. Studying the mechanisms involved in the formation of the OSVZ during embryonic development in the ferret, we have also demonstrated that cells in the OSVZ follow a lineage completely independent from the other germinal layers, a completely unexpected finding, different from what is seen in rodents. In addition, performing in utero electroporation studies in this animal model, we determined that gain of FlnA function severely impairs the integrity of the apical junction belt (which holds together RG cells) and, as a result, most of these cells either delaminate prematurely or die. Overexpression of mutant and wild type PIK3R2 in ferret brain led to a significant increase in Radial Glia Progenitor Cells and Intermediate Progenitor Cells, with concomitant changes in cell cycle parameters as well as accumulation of ectopic neurons in the white matter and deep cortical layers in the juvenile cortex, which in part remained in the mature cortex.
Using intrinsic optical signal (IOS) recording in human brain slices, we demonstrated that seizure onset site was the same in consecutive seizure-like events (SLEs), while investigating the role of lactate for maintenance of ionic homeostasis and synaptic transmission, we found that lactate supports recovery of activity dependent ion concentration changes and synaptic transmission in rat CA3 hippocampus.
In the framework of the clinical study in DS patients, which is ongoing in Workpackage 2, we carried out a preliminary data analysis on the population recruited so far in the retrospective protocol, which include three different studies (historical study, true retrospective study, prospective study). The aim of this analysis was not to produce results that will not be available before completing the recruitment, but to test the system for the final analysis. We concluded that the system is ready and effective for the final analysis to be carried out at the end of the project.
Preliminary data analysis of 41 Dravet patients (historical study) revealed that in this series, there was no correlation between the presence and type of mutation and cognitive outcome. By contrast, the electroclinical phenotype of the first years of life seems to correlate with cognitive outcome: negative prognostic factors are represented by early onset seizures, occurrence of the first seizure in wellness and recurrence of epileptic status.
In the true retrospective study, we analyzed 50 adult patients with a clinical diagnosis of Dravet Syndrome (28 females, 22 males, mean present age of 28 years, ranging from 18 to 50 years of age). All the patients but one have been screened for SCN1A mutation and 94% of them carried a mutation (truncating and missense mutations in 38% and 48% of cases respectively). In 8% of cases, the effect of the mutation was not clear. Epileptic status has been reported in 80% of cases and epilepsy features changed in adulthood. All the adult patients but one are still taking heavy polytherapy in spite of the reduction of seizure frequency. Neurological examination revealed in adults the presence of a variable association of neurological signs: pyramidal and extrapyramidal signs, myoclonus and ataxia. As a matter of fact, in adult population there was a severe impairment of adaptive functioning.
For the LKS/CSWSS Clinical study, we continued to recruit new participants for the multicentre longitudinal study. We enrolled three additional centres in Brussels and London (Great Ormond Street Hospital and St Thomas’ Hospital). And we are in the process of adding four new recruiting sites to the study in addition to the current 14 (2 in the UK - Addenbrookes Hospital, Cambridge; Leicester Royal Infirmary and 2 in the USA - Mayo Clinic, Rochester; Mount Sinai-Beth Israel Medical Center & St. Luke's-Roosevelt Hospital, New York).Our experiments performed for characterizing animal models of Dravet syndrome revealed that the induction of hyperthermic seizures in GEFS+ mice caused a reduction of Calbindin and c-FOS immunoreactivity in the brains of these animals. A 3-day actimeter study on GEFS+ mice showed that seizures induced by hyperthermia (SIH) do not modify the circadian cycle but that the reaction to novelty is significantly increased in the GEFS+ mice with SIH and a working learning/memory is impaired. Using in situ hybridization experiments, we demonstrated that Nav1.1, the protein which is altered in Dravet syndrome, is expressed by CCK positive GABAergic neurons. We performed patch clamp recordings in the CA1 area of the hippocampus showing that CCK+ basket cell interneurons are hyperexcitable in the knock-out Nav1.1+/- model, consistently with a homeostatic response. In addition, we demonstrated that the synaptic release of CCK from CCK+ neurons plays an important role in reducing the decrease of GABAergic inhibition.
In the framework of our studies on Landau-Kleffner syndrome and the CSWS syndrome in animal models, we performed electrophysiological and histological investigations in A/J JAX mice. Electrophysiological experiments in somatosensory and thalamic reticular nucleus slices of A/J JAX mice demonstrated more neuronal excitability compared to control mice. Histological investigations revealed no overt signs of neuro-inflammation, while preliminary stereological cell counts in the thalamic reticular nucleus (RT) indicate an increase of GABAergic cell density in A/J JAX compared to control.
Performing massive parallel sequencing of panels of candidate genes, we demonstrated that defects of the RORB gene cause absence epilepsy. Some of the patients carrying mutations in RORB also experienced focal seizures with centro-temporal spikes, suggesting that mutations in this gene are associated with a wider phenotypic spectrum.
MRI-DTI longitudinal analysis in Grin2a KO mice revealed microstructural anomalies at specific postnatal developmental stages in thalamus and corpus callosum. Preliminary results we obtained through deep cortical recordings of electrical activity in Grin2a KO mice also suggested increased spontaneous cortical activity and altered slow wave activity.
Studying changes in intrinsic excitability following chronic exposure to Charbachol (CCh) in CA3 neurons from rat organotypic slices, we identified a homeostatic response by decreasing the intrinsic excitability of a subset of CA3 neurons caused by the upregulation of Kv7 channels, providing a possible new cellular and molecular target for the treatment of epilepsies.
In the framework of Workpackage 3, we prepared the documentation for the evaluation of the Technical File by the Competent-Body (CB-IMQ), in order to obtain the CE certificate for the HFO prototype developed by Micromed. We also developed a scalp and intracranial HFO protocol using the above prototype.
Comparing MRI images obtained at 7T with those obtained at lower magnetic fields, we assessed an increased diagnostic yield of 7T MRI compared to lower field imaging in cryptogenic patients and in patients with glioneuronal tumors.
Methylation studies performed in Workpackage 4 allowed identifying specific epigenetic signatures (i.e. DNA methylation and histone marks) that distinguish FCDs from other epilepsy samples and FCD subtypes among each other, supporting the histopathological classification. A second round of microRNA profiling in an independent cohort of 20 human surgical brain samples from epilepsy patients with TLE-HS and FCD as underlying pathology, provide first evidence for a microRNA signature specific to FCD type IIb.
Analysis of neurophysiological data for evaluating different types of activity (HFO, spikes, seizures, other oscillatory patterns) as putative markers for epileptic activity associated with focal cortical dysplasia, and comparison with histological correlates in the co-registered surgical specimen (i.e. normal neurons, dysmorphic neurons, astroglia or balloon cells) identified electrophysiological signatures specific for dysmorphic neurons.
Concerning the European Epilepsy Brain Bank (EEBB), we completed data analysis in a total of 9523 patients from 35 European epilepsy centres in 12 European countries. We received additional data in 7286 patients with seizure outcome 12 months after surgery.
Workpackage 5 aims at improving epilepsy treatment in children with FCD ILAE Type II. Out of the 14 participating sites, 5 are open to recruitment and an additional 4 have received on-site training. Plans have been made to deliver all training to sites by the end of 2016 with remaining sites opening early in 2017. The first patient was recruited to the EDIBLE trial on 10th August 2016 by Birmingham Children’s Hospital in the UK. There is continuing monitoring of activities regarding set-up progress, disease prevalence and recruitment rates inform the adjustment to the trial strategy and corrective actions.
In Workpackage 6, aimed at identifying innovative strategies for treatment and prevention of EDD related epilepsy and of its consequences. Within this Workpackage, we developed cell-specific expression of probes that inhibit RE1-silencing transcription factor in neurons and efficiently modulate network excitability in vitro, we converted fibroblasts into functional forebrain GABAergic interneurons by direct genetic reprogramming, to develop a cell therapy approach, and generated and characterized a new rat model of bilateral double cortex with early onset spontaneous epilepsy. We also prepared AAV vectors (AAV-CamKIIa-eGFP-anti Na) that efficiently knockdown the expression of sodium channels in principal glutamaergic neurons.

During the second period, Workpackage 7 mainly dealt with the implementation of the DESIRE dissemination plan. DESIRE was widely represented in and disseminated to the scientific community (52 oral presentations and invited talks, 55 publications and 10 poster acknowledging the EC funding). Information about DESIRE was disseminated to various patients and lay organisations as well as the general public. Furthermore, the project website has been updated, together with the social network profiles. Periodic newsletters are also released. The first release of the exploitation plan has been also performed.
During the second period, Workpackage 8 activities were related to the overall financial and administrative management, including the management of the EC contribution, the preparation and submission of the second periodic report, as well as the set-up of a framework for the third periodic report, the organization of the periodic consortium meetings, including the participation of the Scientific Advisory Committee members and the Ethical Advisory Board.

Potential Impact:
This project gathers a multidisciplinary team of European research groups bridging basic with clinical sciences, which are leading international players in their discipline.
Participating groups have established a strong track record in the multidisciplinary study of MCD, FCD and EE. They have access to advanced experimental models, large patient cohorts, the European Epilepsy Brain Bank, as well as large DNA and cell line collections. They have extensive experience in participation in, and co-ordination of, European projects. This consortium has therefore the potential to make a major impact in diagnosis, genetic counselling, treatment and health care of children with EDD.
The active involvement of patient organizations through the dissemination activities will ensure the rapid implementation of results and immediate benefit for families.
DESIRE will improve the understanding of the etiology and mechanisms of epilepsy and epileptiform disorders. By deciphering PM in EDD and developing mechanism-related, and advanced therapeutic strategies, DESIRE expects to discover novel genes and related molecular pathways that are involved in epilepsy and similar disorders. DESIRE will also help preventing the development of the disease after potentially epileptogenic brain insults.
Identification of novel neurophysiological and neuropsychological biomarkers will lead to earlier diagnosis and treatment, making it possible to prevent or minimize epilepsy related cognitive decline.
DESIRE will also propose the first European and world-wide randomized clinical trial for epilepsy surgery in children with FCD that will address prospectively the true place of surgery in the management of these patients and whether outcome can be improved by utilization of the ketogenic diet prior to surgery.
Up to date, the drug industry has exerted its influence in the domain of epilepsy, neglecting its varied etiologies and consequences and the needs of the pediatric population and considering it as a single entity as far as treatment is concerned.
With most advanced technologies, DESIRE proposes experimental strategies that may rapidly translate into clinical practice by using specific pathophysiological individualized approaches.
The participation of five SMEs operating in the areas of technology and molecular medicine is expected to translate the new technologies into larger scale diagnostic and therapeutic tools and to open new perspectives towards the participation of the industry in an integrated and rational drug discovery process heading towards a more targeted treatment for epilepsy.

List of Websites:

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